Anti-cocaine vaccine

ABSTRACT

An anti-cocaine vaccine employs a cocaine hapten conjugated to a carrier protein. The anti-cocaine vaccine elicits an immune response which reduces the psychoactive effects of cocaine consumption by the production of anti-cocaine polyclonal antibodies. The antibodies may be employed in an ELISA test for assaying cocaine. The immune response elicited by the anti-cocaine vaccine produces antibody producing cells which may be isolated and cloned for producing anti-cocaine monoclonal antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT application serial no.PCT/US96/19982 filed on Dec. 16, 1996, which application is acontinuation in part of application Ser. No. 08/572,849 filed Dec. 14,1995, abandoned the disclosures of which applications are hereinincorporated by reference.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under the NationalInstitute of Drug Abuse grant No. DA 08590. The U.S. government hascertain rights in the invention.

SPECIFICATION

1. Field of Invention

The invention relates to methods for treating cocaine abuse. Moreparticularly, the invention relates to anti-cocaine vaccines that elicitimmune responses for reducing the psychoactive effects of cocaineconsumption.

2. Background

Cocaine is a powerfully addictive substance and new strategies areneeded to treat its abuse. Cocaine degrades spontaneously in vitro andin vivo (E. R. Garrett, et al., Journal of Pharmaceutical Science(1983): vol. 72, p 258-271; D. J. Stewart, et al., Clin. Pharmacol.Ther. (1979): vol. 25, p 464-468). A principle route for the degradationof cocaine is the hydrolysis of the methyl ester to produce thenonpsychoactive compound benzoylecgonine (K. A. Cunningham, et al.,Neuropsychopharmacology (1990): vol 3, p 41-50). Nonspecific esterasesare also known to contribute to the in vivo degradation of cocainethrough cleavage of both the methyl and benzoate esters (M. R.Brzezinski, et al., Biochem. Pharmacol. (1994): vol. 48, p 1747-1755; C.S. Boyer, et al., J. Pharmacol. Exp. Ther. (1992): vol. 260, p 939-946;R. A. Dean, et al. FASEB J. (1991): vol. 5, p 2735-2739; K. Matsubara,et al., Forensic Sci. Intl.(1984): vol. 26, p 169-180; Y. Liu, et al.,J. Chromatography (1982): vol. 248, p 318-320).

Donald Landry, et al. disclose that catalytic monoclonal antibodies(mAbs) directed to the hydrolysis of cocaine can be elicited byimmunization with transition state analogues of cocaine (PCTInternational Application, WO 9320076 A1, published Oct. 14, 1993 basedupon serial number 93-PCT/US 3163, filed Apr. 2, 1993; and Science(1993): vol. 259, p 1899-1901). The catalytic mAbs generated thereby areshown by Landry to catalyze the hydrolysis of cocaine and to reducecocaine levels in human blood thereby. The catalytic mAbs are furtherdisclosed by Landry to be therapeutically employable for treatingcocaine overdose and/or cocaine addiction.

G. P. Basmadjian, et al., disclose that catalytic polyclonal antibodiesdirected to the hydrolysis of cocaine can be elicited by immunizationwith transition state analogues of cocaine (Chem. Pharm. Bull. (1995):vol. 43, p 1902-1911). Preliminary results showed that mice immunizedwith immunoconjugates derived from these analogues produced, in somecases, high titers of serum catalytic antibodies as judged from an invitro radioassay. No further work has been reported.

S. Spector, et al. disclose that mice can be actively immunized with amorphine immunogen and that serum from such mice contain antibodies thatbind dihydromorphine. Morphine effects and the plasma concentration ofmorphine were shown to be diminished in these immunized mice (S.Spector, et al., Pharmacol. Rev. (1973): vol. 25, p 281-291; and B.Berkowitz, et al., Science (1972): vol. 178, p 1290-1292).

What is needed is an anti-cocaine vaccine for generating an activeimmunization to cocaine. More particularly, the antibodies generated bythe anti-cocaine vaccine should block the actions of the cocaine bypreventing the entry of cocaine into the central nervous system. Theanti-cocaine vaccine should be characterized by reduced side effects ascompared to the side effects associated with treatments based onmanipulation of central neurotransmitter function.

SUMMARY OF THE INVENTION

It is disclosed herein that generating an active immunization to cocaineoffers a means of blocking the actions of the drug by preventing it fromentering the central nervous system. This method of treatment has lessside effects than treatments based on manipulation of centralneurotransmitter function. The design and preparation of a cocaineimmunogen requires special regard for the stability of cocaine both freeand as a haptenic determinant. Immunochemistry and a well-definedbehavioral paradigm are brought together to address the problem ofinactivation of the psychostimulant actions of cocaine. Activeimmunization is achieved with a novel, stable cocaine conjugate,disclosed below, which suppresses locomotor activity and stereotypedbehavior in subjects induced by cocaine but not by amphetamine.Moreover, following acute injection of cocaine, levels of cocaine in thestriatum and cerebellum of the immunized subjects are significantlylower than those of control animals. These results demonstrate thatimmunopharmacotherapy can be employed for treating cocaine abuse.

The design and preparation of a cocaine immunogen requires specialattention to the stability of free cocaine in solution and as a haptenicdeterminant. Cocaine degrades spontaneously in vitro and in vivo. (E. R.Garrett, et al., J. Pharmaceutical Sci. (1983): vol. 72, p 258-271; andD. J. Stewart, et al., Clin. Pharmacol. Ther. (1979): vol. 25, p464-468. Degradation occurs largely through hydrolysis of the methylester to produce the nonpsychoactive compound benzoylecgonine (K. A.Cunningham, et. al., Neuropsychopharmacol.(1990): vol. 3, p 41-50).Nonspecific esterases are also known to contribute to the in vivodegradation of cocaine through cleavage of both the methyl and benzoateesters (M. R. Brzezinski, et. al., Biochem. Pharmacol. (1994): vol. 48,p 1747-1755; C. S. Boyer, et. al., J. Pharmacol. Exp. Ther. (1992): vol.260, p 939-946; R. A. Dean, et. al. FASEB J. (1991): vol. 5, p2735-2739; K. Matsubara, et al., Forensic Sci. Intl.(1984): vol. 26, p169-180; Y. Liu, et. al., J. Chromatography (1982): vol. 248, p318-320). Conjugates that display epitopes structurally similar to thoseof metabolites, especially benzoylecgonine, would compromise the avidityand specificity of a cocaine-specific immune response (M. J. Taussig,Current Topics Microbiol. Immunol. (1973): vol. 60, p 125-174; and A. L.Misra, et al., Res. Comm. Che. Path. Pharmacol. (1976): vol. 13, p579-584). Also, an appreciable benzoylecgonine titer would beexceptionally detrimental since the antiserum would be inadequate inneutralizing cocaine, particularly in the presence of rapidly formed andstable metabolites. Although each retains the phenyl ring as a majorrecognition element, the neutrality of cocaine contrasts with thenegatively charged benzoylecgonine, a factor in antibody binding, makingit possible to maximize the affinity and selectivity for cocaine. Byjoining the carrier protein to the cocaine framework using a linker atthe position occupied by the methyl ester, any minor decomposition ofthe linked hapten results not in a benzoylecgonine response, butprimarily in nonhaptenic recognition. Attention to such aspects of theimmunochemistry must be emphasized in view of an unsuccessful report ofa potential cocaine prophylactic (O. Bagasra, et al.,Immunopharmacol.(1992): vol. 23, p 173-179; G. Gallacher,Immunopharmacol (1994): vol. 27, p 79-81). The hapten 4 (compound 4) wassynthesized in four steps starting from (−)-cocaine (FIG. 1). The keyreaction, alkylation of (−)-ecgonine, introduced the required tether.The stereochemical configuration remained intact at C-2 of the tropanenucleus. This ester linker mimics the alkyl character of the methylester of cocaine which is important for recognition of this part of themolecule. Coupling of 4 to keyhole limpet hemocyanin (KLH) afforded theconjugate, 4-KLH, for immunization.

One aspect of the invention is directed to cocaine analogs. Preferredcocaine analogs are represented by the following structures:

where n is greater than or equal to 2 and less than or equal to 8. Inpreferred embodiments, n is greater than or equal to 4, less than orequal to 6, or equal to five.

Another aspect of the invention is directed to cocaine immunoconjugates.Preferred cocaine immunoconjugates are represented by the followingstructure:

where n is greater than or equal to 2 and less than or equal to 8. Inpreferred embodiments, n is greater than or equal to 4, less than orequal to 6, or equal to five. An additional preferred cocaineimmunoconjugate is represented by the following structure:

wherein n and m are greater than or equal to 4, less than or equal to 6,or n is six and m is four.

Another aspect of the invention is directed to a method for suppressingpsychoactive effects of cocaine within a subject. The method includes astep wherein an anti-cocaine vaccine is administered to the subject. Theanti-cocaine vaccine is of a type which includes an injectable sterilesolvent and an immunogenic amount of a cocaine immunoconjugate.Preferred immunoconjugates are indicated above.

Another aspect of the invention is directed to an anti-cocaine vaccine.The anti-cocaine vaccine comprises a sterile injectable medium and acocaine immunoconjugate admixed with said sterile injectable medium.Preferred immunoconjugates are indicated above.

Another aspect of the invention is directed to a method for reducingpsychoactive effects displayed by a subject upon administration ofcocaine. The method comprises two steps. In the first step, ananti-cocaine immune response is elicited within the subject byvaccination with an anti-cocaine vaccine. The anti-cocaine vaccineincludes one of the cocaine immunoconjugates indicated above. In thesecond step, cocaine is administered to the subject.

Another aspect of the invention is directed to a method for obtaininganti-cocaine polyclonal antibodies. The method includes two steps, ananti-cocaine immune response is elecited within a subject by vaccinationwith an anti-cocaine vaccine. The anti-cocaine vaccine includes one ofthe cocaine immunoconjugates indicated above. In the second step,anti-cocaine polyclonal antibodies are isolated from the subject.

Another aspect of the invention is directed to anti-cocaine polyclonalantibodies produced according to the method indicated above.

An other apsect of the invention is directed to a method for obtaininganti-cocaine monoclonal antibodies. The method includes three steps. Inthe first step, an anti-cocaine immune response is elicited within asubject by vaccination with an anti-cocaine vaccine. The anti-cocainevaccine includes one of the cocaine immunoconjugates indicated above. Inthe second step, an antibody producing cell from the subject of saidStep A which expresses an anti-cocaine antibody is isolated and cloned.In the third step, anti-cocaine monoclonal antibody expressed byantibody producing cell isolated and cloned in the second step areisolated.

Another aspect of the invention is directed to anti-cocaine monoclonalantibodies produced according to the method indicated above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a synthetic scheme for making the cocaine hapten 4employed in the anti-cocaine vaccine.

FIG. 2 illustrates analysis of the serum antibodies generated byadministration of the anti-cocaine vaccine.

FIG. 3 illustrates the alteration of psychoactive effects resulting fromadministration of the anti-cocaine vaccine in subjects as compared tosubjects not having received the vaccine.

FIG. 4 illustrates the effects of the anti-cocaine vaccine upon levelsof cocaine found in the brain of subjects.

FIG. 5 illustrates the synthesis of hapten 7 using the following steps;top scheme: a) Br(CH₂)₅CO₂Bn, NaOH, pyridine; b) (i) LDA, (ii) add 8; c)H₂, Pd/C; lower scheme illustrates the synthesis of compound 8 using thefollowing steps: a) benzyl alcohol, NEt₃; b) PCl₅.

FIG. 6 illustrates the synthesis of hapten 15 using the following steps:a) (i) trichloroethyl chloroformate, (ii) Zn, formic acid; b) 1.25 MHCl, reflux; c) MeOH, HCl; d) Br(CH₂)₃CO₂Bn, NEt₃; e) (i) LDA, (ii) add8; f) H₂, Pd/C.

FIG. 7 illustrates the synthesis of hapten 18 using the following steps;top scheme: a) (i) MeOH, HCl, (ii) free-base; b) (i) LDA, (ii) add 19;c) H₂, Pd/C; lower scheme illustrates the synthesis of compound 19 usingthe following steps: a) P(OMe)₃, light; b) (i) NaH, (ii) Br(CH₂)₃CO₂Bn;c) (i) TMSBr, (ii) oxalyl chloride, (iii) benzyl alcohol, NEt₃; d) PCl₅.

FIG. 8 illustrates the synthesis of hapten 21 using the following steps;top scheme: a) Br(CH₂)₅CO₂Bn, NaOH, pyridine; b) (i) LDA, then 22; c)H₂, Pd/C; lower scheme illustrates the synthesis of compound 22 usingthe following steps: a) dibenzyl phosphite, NaH; b) PCl5.

FIG. 9 illustrates the synthesis of hapten 24 using the following steps;top scheme: a) Br(CH₂)₅CO₂Bn, NaOH, pyridine; b) (i) LDA, (ii) add 25;c) H₂, Pd/C; lower scheme illustrates the synthesis of compound 25 usingthe following steps: a) trifluoroethanol, NEt₃.

FIG. 10 illustrates the synthesis of hapten 30 using the followingsteps; top scheme: a) (i) benzyl bromide, Me₄NOH, MeOH, DMF, (ii)free-base; b) add 31, NEt₃; c) H₂, Pd/C; d) methylamine, EDC, DMF; e)trifluoroacetic acid; lower scheme illustrates the synthesis of compound31 using the following steps: a) Br(CH₂)₅CO₂t-butyl, NaH; b) diluteNaOH; c) oxalyl chloride.

FIG. 11 illustrates the synthesis of hapten 37 using the followingsteps: a) (i) isobutylene, H₂SO₄, (ii) free base; b) Br(CH₂)₅CO₂Bn,NEt₃; c) benzoyl chloride, NEt₃, DMAP; d) trifluoroacetic acid; e)methylamine, EDC; f) H₂, Pd/C.

FIG. 12 illustrates the synthesis of hapten 43 using the followingsteps: a) NH₄OAc, HOAc, benzene, reflux; b) (i) NaCNBH₃, pH 4, MeOH, RT,(ii) benzoyl chloride, NaHCO₃, dioxane-H₂O, (iii) separation of isomers;c) H₂O, reflux; d) (i) H₂N(CH₂)₅CO₂Bn tosylate, EDC, NEt₃, DMAP, (ii)separation of isomers; e) H₂, Pd/C, MeOH; f) (i) Sufosuccinimide,EDC-HCl, r.t. (ii) KLH or BSA, r.t.

FIG. 13 illustrates the general structures of haptens (left column) andconjugates (right column).

DETAILED DESCRIPTION Conjugation of Cocaine Hapten with Carrier

The immunoconjugate 5 used in the anti-cocaine vaccine was prepared bycoupling the hapten 4 with a carrier as described as follows:

Hapten 4 (20 mmol) was activated for coupling in dimethylformamide (DMF)(200 μl) using an aqueous solution of1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC) (26 μmol) andN-hydroxysulfo-succinimide (sulfo-NHS) (26 μmol) (Pierce). The aqueouscontent was 10%. After 20 hours at 22° C. the solution was added to KLH(20 mg) in 4 ml of 50 mM phosphate buffer (PB), pH 7.5. This was kept at4° C. for 20 hours during which time turbidity developed. The finesuspension was dialyzed against two changes of 100 mM PB, pH 7.0, inwhich it could be frozen and stored for at least two years. Propionicacid was activated and coupled in an identical fashion. This conjugateserved as the KLH control for immunization. The same protocol was usedto couple 4 to bovine serum albumin (BSA) affording a nonturbid solutionof the conjugate used for ELISA. In addition, both KLH and BSA weremodified as above with (carbonyl-14C-benzoyl)-4 to determine the extentof labeling and stability of the conjugate. The number of ligands were29 and 19, respectively. Both conjugates were completely stable in 100mM PB, pH 7.4, 22° C. for at least 48 hours.

Competitive binding studies demonstrated that the immune response washighly specific for cocaine. The discrimination was greater than900-fold versus benzoylecgonine and there was no detectable inhibitionof cocaine-binding by ecgonine methyl ester. Consequently, inhibition ofcocaine-binding immunoglobulins would not be expected in vivo from thesemetabolites. The polyclonal antibodies from, such an immune responsehave also been isolated and purified from serum using standardtechniques (E. Harlow and D. Lane, Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory, New York, 1988). These immunoglobulinswere found to have cocaine-binding characteristics similar to the serumitself.

The KLH immunoconjugates prepared from haptens 4 and 43 were also usedto obtain monoclonal antibodies (mAbs) with high affinity andspecificity for cocaine. These mAbs were derived from murine subjectsaccording to well-established procedures (E. Harlow and D. Lane,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory, NewYork, 1988); Peters, J. H. and Baumgarten, H., Eds., MonoclonalAntibodies, Springer-Verlag, New York, 1992). A number of mAbs werefound to have binding constants in the submicromolar range and were10-1000 times more specific for cocaine versus cocaine metabolites.

In further studies, immunoconjugates prepared from haptens 4 and 43 haveelicited a competent immune response in murine subjects using differentcarrier proteins (KLH, BSA) and adjuvants (RIBI, aluminum hydroxide). Anumber of other methods would likely be feasible.

Carrier Proteins

A small haptenic molecule must first be conjugated to an immunogeniccarrier, such as a protein, in order to elicit a competent immuneresponse (Williams, C. A. and Chase, M. W., Eds., Methods in Immunologyand Immunochemistry (1967): vol. 1, pp 120-187; G. T. Hermanson,Bioconjugate Techniques, (Academic Press, New York, 1996), pp 419-455).There are several different carrier proteins that can be used forcoupling to haptens. The two most commonly used carrier proteins arekeyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Both ofthese proteins work well and each has particular utility. KLH, due toits structural features and large size, is highly immunogenic. Inaddition, KLH generally forms particulate immunoconjugates that mayfurther enhance immunogenicity, but also makes such conjugates lesssuitable for enzyme-linked immunosorbent assay (ELISA). Hence, it isoften the protein of choice for generating an anti-hapten immuneresponse. On the contrary, BSA immunoconjugates are usually soluble andtherefore very useful for ELISA. However, BSA conjugates have also beenused for immunization in conjunction with a different solubleimmunoconjugate that is used in subsequent ELISA screening procedures.Other carrier proteins that are often used for coupling to haptens andfind utility for either immunization or ELISA include ovalbumin, rabbitserum albumin, and thyroglobulin. Alternative carriers usually reservedonly for immunization purposes include toxoids derived from diphtheriaand tetanus.

Coupling of Carrier Proteins to Haptens

The following conditions are exemplary for the coupling of haptens tocarrier proteins and are included to illustrate one of manypossibilities for coupling of the indicated haptens with carrierproteins. Other activated esters in lieu of (EDC) may be used andinclude dicyclohexylcarbodiimide (DCC) and 2,5,6-Cl₃(C₆H₂)COCl(Aldrich). Other solvents in lieu of DMF may be used and includeacetonitrile, methylene chloride, chloroform, ethylacetate andtetrahydrofuran. Other carrier proteins that are often used for couplingto haptens include ovalbumin, rabbit serum albumin, thyroglobulin andtoxoids derived from diphtheria and tetanus. Various pH buffer systemscan be used and temperatures and reaction times may vary as indicated,depending upon the hapten and carrier protein combination.

Representative conditions are follows: Hapten 4, 7, 15, 18, 21, 24, 30,37 or 43 (20 mmol) was activated for coupling in dimethylformamide (DMF;other possible solvents listed above) (200 μl) using an aqueous solutionof 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC other possibleactivated ester reagents listed above) (26 μmol) andN-hydroxysulfo-succinimide (sulfo-NHS) (26 μmol) (Pierce; reagentincreases yields, but not necessary). The aqueous content was 10% (range5-15%). After 20 hours at 22° C. (range 15-30° C.) the solution wasadded to KLH, (20 mg; other possible carrier proteins listed above) in 4ml of 50 mM phosphate buffer (PB; other buffer systems in same pH rangemay be used), pH 7.5 (pH range 6.5-8.5). This was kept at 4° C. (range0° C. to 10° C.) for 20 hours during which time turbidity developed. Thefine suspension was dialyzed against two changes of 100 mM PB, pH 7.0,in which it could be frozen and stored for at least two years.

Adjuvants

Despite their large size and multiple epitopes, isolated proteinstructures, such as immunoconjugates, generally require injection aspart of an adjuvant medium in order to elicit a competent primary immuneresponse (Williams, C. A. and Chase, M. W., Eds., Methods in Immunologyand Immunochemistry (1967): vol 1, pp 197-209; E. Harlow and D. Lane,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory, NewYork, 1988), pp 96-97; Peters, J. H. and Baumgarten, H., Eds.,Monoclonal Antibodies, (Springer-Verlag, New York, 1992), pp 58-60).Adjuvants induce potent cellular and humoral immune responses to avariety of antigens including carbohydrates, peptides, and proteins andhave been valuable in the production of polyclonal and monoclonalantibodies. Freund's adjuvants (water-in-oil emulsions with or withoutheat-inactivated Mycobacterium tuberculosum) has been the most employedover the years. Recently, the RIBI adjuvant system (RAS) (RIBIImmunochem Research, Inc.) has found widespread use. Anothercommercially available adjuvant is based on aluminum hydroxide. Such anadjuvant may be most suitable for human use. In this regard, newermethods for achieving adjuvant effects have incorporated antigens intobiologically degradable polymers and liposomes (J. Kohn, et al., J.Immunol. Methods (1986): vol. 95, p 31-38; D. Davis, et al., Immunol.Lett. (1983): vol. 14, p 341-348).

Assessment of Efficacy

To assess the efficacy of immunization, the psychostimulant effects ofcocaine were measured in the rat. This psychostimulant effect is adose-dependent increase in locomotor activity and stereotyped behaviorbelieved to result from cocaine's actions on dopaminergic neurons in theventral forebrain and striatum (P. H. Kelly, et al. Eur. J. Pharmacol.(1976): vol. 40,p 45-56.). Male Wistar rats were first tested inphotocell cages after treatment with intraperitoneal (i.p.) cocaine-HCl(15 mg/kg) to determine pre-immunization drug response (baseline). Thisdose of cocaine is an intermediate dose that produces a significantlocomotor activation and modest stereotyped behavior. Lower dosesproduce less locomotor activity and virtually no stereotyped behavior.Higher doses also produce less locomotor activity but more robuststereotyped behavior (M. Lyon, et al., in Current Developments inPsychopharmacology, Essman, W. & Valzelli, L. Eds., (Spectrum, New York,1975), vol. 2, pp 89-163.). Experimental animals were injected threedays later with 4-KLH i.p. as an emulsion in RIBI adjuvant and controlanimals treated with an emulsion containing only KLH. This primaryinoculation was followed by boosts at 21 and 35 days. No stimulation ofbehavioral activity or other behavioral effects were observed followingeach immunization, consistent with the inability of the conjugate tocross the blood-brain barrier. Serum samples were assayed 7 days aftereach boost. Titers were typically 1:24,000 by enzyme-linkedimmunosorbent assay (ELISA) and 1:50 in a solution radioimmunoassay, asillustrated in FIG. 2. Following the final boost of 4-KLH or KLH, theanimals were challenged with systemic cocaine and their locomotorresponses and stereotyped behavior were measured (first cocainechallenge). The ambulatory response (crossovers) to cocaine wassignificantly different between control and immunized animals, asillustrated in FIG. 3. This measure was 42% lower in the experimentalgroup compared to baseline, while the control group displayed a 30%increase compared to baseline. A similar decrease in the psychostimulantactions of cocaine was seen in immunized animals upon two subsequentcocaine challenges with differences in locomotion between groupssignificant at the time of the first and second challenges, but not thelast, as illustrated in FIG. 3. Stereotyped behavior was suppressed inexperimental animals consistent with the decreases in locomotor activityand this effect was significant across all challenges, as illustrated inFIG. 3. The antibody titer, as measured seven days prior to the finalinoculation, was essentially unchanged eight days following the lastchallenge.

Analysis of serum immunoglobulin dissociation kinetics, antigen-bindingcapacity and the effect of antigen dilution (P. Minden, et al., inHandbook of Experimental Immunology. Weir, D. M., Ed., (Davis Co.,Philadelphia, 1967), chap. 13, pp. 463-492; M. W. Steward et al.,Antibody Affinity: Thermodynamic Aspects and Biological Significance,(CRC Press, Boca Raton, Fla., 1983); H. N. Eisen, in Methods in MedicalResearch. Eisen, H. N., Ed., (Yearbook Medical Publishers, Chicago,1964), pp. 106-114; J. H. Hill, et al. Clin. Exp. Immunol. (1973): vol.15, p 213-224.) were consistent with an in vivo antibody excess having amicromolar average binding constant. In addition, comparison with apurified anti-cocaine murine mAb indicated that one milliliter ofundiluted serum from an immunized rat could bind the same amount ofcocaine as 4 mg/ml of monoclonal antibody. To complement theseexperiments, plasma cocaine levels were determined by HPLC according toa modified method of Benuck (M. Benuck, et al., J. Pharmacol. Exp.Ther.(1987): vol. 243, p 144-149) following a 15 mg/kg i.p. injection ina separate group of animals. A measured peak concentration of 5.56±0.31μM occurred at 5 minutes which decayed with a half-life of 25 minutes.Taken together, the data suggest that at equilibrium at least 50% of thecocaine present in the blood under physiological conditions is likely tobe bound.

To support the hypothesis that the observed psychomotor suppression wasspecific to cocaine, a second group of animals was immunized asdescribed above, then challenged with amphetamine (0.75 mg/kg) i.p.three days after the last boost. Amphetamine stimulates locomotoractivity by facilitating the release of dopamine and, like cocaine, byblocking dopamine uptake. Amphetamine-induced locomotor activity was notsignificantly different between groups, viz. in one sample, the totalcrossover counts for controls was 892.35±177.51 while the total forexperimentals was 948.75±217.51; F(1,12)<1. When these animals weresubsequently challenged with cocaine (10 days after the last boost),both measures of locomotor activity were again suppressed inexperimental animals as compared with controls, viz., F(1,12)=14.8,P<0.002. To investigate the basis for this blunted behavioral response,animals were sacrificed thirty minutes after receiving the cocaineinjection and their brains extracted for analysis. Levels of cocainewere found to be 52% lower in the striatal tissue and 77% lower incerebellar tissue of the immunized animals compared to controls, asillustrated in FIG. 4.

In the first group of animals, the reduced psychomotor response observedduring the first challenge persisted following subsequent cocaineinjections in the experimental group, as illustrated in FIG. 3. At thesecond cocaine challenge, locomotor activity and stereotyped behaviorwere significantly decreased in the experimental group as shown in FIG.3. At the third cocaine challenge, locomotor activity did not differsignificantly between the control and experimental groups. However,there continued to be a highly significant difference in the amount ofstereotyped behavior. The decrease in stereotyped behavior but notlocomotor activity compared to controls may reflect the change inprofile of psychomotor stimulation associated with repeated cocaineexposure. Typically, in control animals, repeated administration ofcocaine shifts the cocaine dose response to the left (sensitization) andthis is reflected in less locomotor activity but more stereotypedbehavior (M. Lyon, et al., in Current Developments inPsychopharmacology, Essman, W. and Valzelli, L. Eds., (Spectrum, NewYork, 1975), vol. 2, pp. 89-163). There was some evidence of anincreased psychomotor response and a prolonged stereotyped behavioralresponse in the control group but this sensitization did not occur inthe immunized animals, as illustrated in FIG. 3. However, it isimportant to emphasize that the overall psychomotor response to cocaineremained dramatically blunted in the immunized animals. These findingssuggest that the level of cocaine reaching critical sites of action inthe central nervous system of immunized rats was decreased at the timeof post-immunization challenges, thus producing a markedly diminishedpsychomotor effect characteristic of lower doses of cocaine (P. H.Kelly, et al. Eur. J. Pharmacol. (1976): vol. 40,p 45-56). Thishypothesis is supported by the maintenance of antibody titer which wasthe same before the first cocaine challenge and 8 days after the lastcocaine challenge, a span of 25 days. Given enough time between cocaineinjections, saturation of the immune mechanism may be avoided andprotection may be sustained.

Analysis of cocaine concentrations in cerebral tissue revealedconsiderably lower levels of cocaine in the brains of immunized animalscompared to those of controls after cocaine injection. This finding iscompatible with the diminished psychostimulant activity observed in theimmunized group. Moreover, there is a good correlation between theestimated levels of bound cocaine in the bloodstream and the differencefound in the brains of experimental and control animals. In this regard,it seems that extreme antibody affinities are not necessary, but onlythat they match the peak concentrations of cocaine. It is possible thatthe thermodynamics of interaction between cocaine binding to antibodiesand receptors may be at an optimum (M. C. Ritz, et al., Science (1987):vol. 237, p 1219-1223; M. Fischman, et al., Pharmacol. Biochem. Behav.(1983): vol. 18, p 123-127). In this case, the in vivo immunoglobulinconcentration can be a controlling factor in neutralizing the action ofthe drug. Importantly, the results are an indication that modulation ofcocaine levels in the circulation directly influence behavior. This mayhave implications regarding human administration in that peak plasmaconcentrations parallel maximum subjective effects (J.Javaid, et al.,Science (1978): vol. 202, p 227-228). However, the effects of this typeof vaccination protocol on the human condition (i.e. “binge-like”patterns) are difficult to estimate at this time and will requireadditional study.

Accordingly, it is disclosed herein that the immune-mediated responsecan be employed to alter reinforcing actions of cocaine in the contextof drug dependence subjects. Because immunization therapy would exertits actions outside of the central nervous system, it has none of theside effects of conventional pharmacotherapy. In addition, the protocoldisclosed herein can be implemented as a prophylactic treatment and inrelapse prevention. Thus, immunopharmacotherapy offers a nontoxic,substance-specific strategy for cocaine abuse treatment that does notaffect normal neurochemical physiology.

Analysis of Immune Response

After vaccination with the anti-cocaine vaccine, serum from immunizedsubjects was tested for production of anti-cocaine antibodies. FIG. 2illustrates ELISA and radioimmunoassay (see inset) titer measurements ofserum from rats immunized with 4-KLH. ELISA plates (96-well) (costar3590) containing 2.75 ng/well 4-BSA in 50 μl of 10 mM phosphatebuffer/150 mM NaCl (PBS), pH 7.4 were dried overnight at 37° C. This wasfollowed by routine methanol fixing and blocking with blotto (nonfatmilk) in PBS. Rat serum was serially diluted beginning with a 1:200dilution in blotto. Primary antibody (serum) binding was allowed to takeplace for 1 hour in a moist chamber at 37° C. After washing, 200 ng/wellgoat anti-rat IgG conjugated with alkaline phosphatase (SouthernBiotechnologies Associates, Inc.) in 50 μl PBS-blotto was added andincubated for 1 hour in a moist chamber at 37° C. The plates werethoroughly washed with water, air dried and developed by adding 100μl/well of 200 μM p-nitrophenylphosphate (Pierce) in 100 mM4-morpholinepropanesulfonic acid (MOPS), pH 7.4. After 3 hours at roomtemperature, the absorbance was measured at 405 nm in a microplatereader (Molecular Dynamics). Radioimmunoassay was conducted in PBS, pH7.4 in the presence of 5% dimethylsulfoxide (DMSO), 22° C. [3H]-cocaine(specific activity=0.234 Ci/mmol, prepared from norcocaine and[3H]-methyl iodide from Amersham), to give a final concentration of 0.33μM, was added to varying dilutions of rat serum, capable of binding morethan and less than 50% of the offered counts. The samples were thentreated as in the following references, viz. (P. Minden, et al. inHandbook of Experimental Immunology, Weir, D. M. Ed.(Davis Co.,Philadelphia, 1967); M. W. Steward, et al., Antibody Affinity:Thermodynamic Aspects and Biological Significance, (CRC Press, BocaRaton, Fla., 1983); H. N. Eisen, in Methods in Medical Research, Eisen,H. N. Ed., (Year Book Medical Publishers, Chicago, 1964); J. H. Hill, etal., Clin. Exp. Immunol. (1973): vol. 15, p 213-224).

Analysis of Psychoactive Effects

An analysis of the psychoactive effects of cocaine administration torats immunized with the anti-cocaine vaccine is illustrated in FIG. 3.Locomotor activity (crossovers) and stereotyped behavior (sniffing andrearing) following intraperitoneal injection of cocainepost-immunization. Immunizations consisted of a 400 μl bolusintraperitoneal (i.p.) injection of a RIBI adjuvant (MPL®+TDM) (RIBIImmunochem Research, Inc.) containing approximately 250 μg 4-KLH or KLHin 100 mM, PB, pH 7.4. The last boost was administered without adjuvant.The figure shows the response to post-immunization cocaine challenge onthe third (A), seventh (B), and tenth (C) day following the lastimmunization boost. Locomotor activity was measured in photocell cagesas in described by Gold (L. H. Gold, et al., Pharmacol. Biochem. Behav.(1988): vol. 29, p 645-648). After a 90 minute period of habituation,animals received an i.p. injection of 15 mg/kg cocaine HCl mixed insaline solution (bolus 1 ml/kg) and their locomotor responses measuredduring a 90 minute session. Based on locomotor activity scores, animalswere assigned to the experimental or control group in ranking order.Locomotor data were analyzed by subjecting ten minute total means forlocomotor activity to a two-factor analysis of variance (ANOVA)(group×time) with repeated measures on the within-group factor, time.Stereotyped behavior was rated as previously described (J. Fray P. J. etal., Psychopharm. (1980): vol. 69, p 253-259. Data was analyzed by alikelihood ratio method, the “Information statistic” (T. W. Robbins, inHandbook of Psychopharmacology, Iversen, L., Iversen, S. and Snyder, S.,Eds., (Plenum, New York, 1977), pp. 37-82; S. Kullback, InformationTheory and Statistics, Dover Press, New York, 1968). These figuresdepict the pooled data from two identical studies for a total of (n=16).At the time of the first cocaine challenge, there was a significantdifference between control and experimental groups in both meanactivity, viz. (A, top) (control: 987.44±149.5; experimental:555.07±124.7) F(1,30)=4.13; and stereotypy, viz. (A, bottom) 2Î=85.25,df=1, 9. Both psychomotor measures were again significantly different atthe time of the second cocaine challenge, viz. locomotor (B, top)(control: 1035.19±152.92; experimental: 598.25±121.46) F(1,30)=3.97, andstereotopy, viz. (B, bottom) 2Î=94.12, df=1,9. By the last cocainechallenge, locomotor activity did not differ significantly betweengroups, viz. (C, top) (control:

719.23±133.77; experimental: 443.24±199.50), whereas there was asignificant difference in stereotypy, viz. (C, bottom) 2Î=85.25, df=1,9; * P<0.05, control group different from experimental group.

Measurement of Cocaine Levels in the Brain

FIG. 4 illustrates the measurement of cocaine levels in the brain. Braincocaine levels 30 minutes after i.p. cocaine injection. Cocaineconcentrations were measured by reverse-phase HPLC as described byBenuck (M. Benuck, et al., J. Pharmacol. Exp. Ther.(1987): vol. 243, p144-149) on brain tissue from animals tested as before (10 days afterlast boost) except that locomotor activity was tested for only 30minutes post-injection, at which time they were killed by decapitation.Their brains were rapidly removed and the striata and cerebellumdissected as previously described by Kelly (P. H. Kelly, et al., BrainResearch (1975): vol. 94, p 507-522). Immediately following dissection,the striata and cerebellar samples from each animal were weighed, thenfrozen at −70∞ C. for three days prior to cocaine extraction. Cocaineand its metabolites were extracted from brain tissue as described byBenuck, viz. (M. Benuck, et al., J. Pharmacol. Exp. Ther.(1987): vol.243, p 144-149). Briefly, approximately 210 mg tissue was sonicated with300 ml acetonitrile then centrifuged. The supernatant was decanted andcocaine and its metabolites were extracted into 700 mlchloroform/ethanol (4:1) and 70 ml of 0.1 M NaHCO3. Appropriate tissuesamples for standards were prepared and analyzed according to the methodof Benuck, viz. (Benuck, M., et al., J. Pharmacol. Exp. Ther.(1987):vol. 243, p 144-149). The cocaine levels measured correspond to freecocaine because cocaine-binding antibodies do not cross the blood brainbarrier, as evidenced by chromatographic separation and ultravioletabsorption spectra. Data were analyzed by one factor ANOVA. (A) Striatalcocaine concentrations in experimental animals were significantly lowerthan those of control animals, viz., F(1,12)=10.37; P<0.01. (B)Cerebellar tissue was sampled to test the generality of cocainedistribution. Cerebellar cocaine concentrations in experimental animalswere significantly lower than those of control animals, viz.F(1,12)=7.30; P<0.05; *P<0.05. Data from two animals in the controlgroup were not included in the cocaine-locomotor analysis due to twoinadequate cocaine injections which resulted in a seizure (n=1), a limphind-limb (n=1). Final group size: control (n=6), experimental (n=8).

Production of Monoclonal Antibodies

The anti-cocaine monoclonal antibodies of the present invention aretypically produced by immunizing or vaccinating a subject with aninoculum of an anti-cocaine vaccine having a cocaine immunoconjugate asdescribed herein. An anti-cocaine immune response is thus elicited orinduced in the subject with the generation of anti-cocaine antibodies.The preparation of antibodies by vaccination with antigenic immunogensis well known in the art and is described in U.S. Pat. Nos. 5,279,956,5,306,620 and 5,321,123, the disclosures of which are herebyincorporated by reference.

Isolation and cloning of the anti-cocaine antibody producing cells thatare generated following immunization procedures described above areachieved in a variety of ways including the preparation of hybridomas asfirst described by Kohler and Milstein, Nature, 256:495-497 (1975), andfurther described in U.S. Pat. Nos. 5,279,956, 5,306,620 and 5,321,123,the disclosures of which are hereby incorporated by reference.Alternative exemplary approaches to isolate and clone an anti-cocaineantibody producing cell of the present invention is achieved through theuse of recombinant DNA technology with immunological repertoires asdescribed by Orlandi et al., Proc. Natl. Acad. Sci., USA, 86:3833-3837(1989), Sastry et al., Proc. Natl. Acad. Sci. USA, 86:5728-5732 (1989),Huse et al., Science, 246:1275-1281 (1981) and U.S. Pat. Nos. 4,683,202and 5,427,908, the disclosures of which are hereby incorporated byreference. The above-identified references also describe exemplarymethods for isolating an anti-cocaine monoclonal antibody expressed byan anti-cocaine antibody producing cell obtained from either classicalhybridoma methodologies or by recombinant means. Other methods forobtaining anti-monoclonal antibodies of this invention are also wellknown in the art.

Synthetic Protocols

The preferred synthetic route for the total synthesis of hapten 4 isillustrated in FIG. 1. Synthesis of hapten 4 involves synthesizing thefollowing intermediates, viz. compounds 1, 2, and 3.

Another aspect of the invention is directed to anti-cocaine vaccineshaving cocaine immunoconjugates with alternative cocaine haptens, viz.compounds 7, 15, 18, 21, 24, 30, 37, and 43. Each of the alternativecocaine haptens is transformed to its corresponding immunoconjugate byan amide linkage between its linker group and a carrier, as disclosedfor immunoconjugate 4.

FIGS. 1 and 5-12 and the protocols below disclose the synthesis of allhaptens.

Synthesis of Compound 1,[1R-(exo,exo)]-3-hydroxy-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylicacid (common name: (−)-ecgonine), as illustrated in FIG. 1

Compound 1. Commercially available (−)-cocaine (Sigma Chemical Co.) wasdissolved in 1.25 M HCl (1 g cocaine/12 ml). The mixture was allowed tostir for 16 hours at 110-115° C. (gentle reflux). After cooling to roomtemperature, the mixture was thoroughly extracted with ether to removethe benzoic acid by-product. The acidic aqueous layer was evaporated ona rotary evaporator under high vacuum and then the residue wasredissolved in water and lyophilized to afford a white solid thatrequired no further purification (92% yield).

Synthesis of Compound 2,[1R-(exo,exo)]-6-[[[3-hydroxy-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]carbonyl]oxy]-hexanoicacid phenylmethyl ester, as illustrated in FIG. 1

Compound 2. Compound 1 (2.0 g, 9.0 mmol) was suspended in a mixture ofpyridine (250 ml, 3.1 mmol), finely powdered NaOH (760 mg, 19 mmol) andbenzyl 6-bromohexanoate (7.8 g, 27 mmol) (synthesized as describedbelow). The mixture was heated to 80° C. and allowed to stir for 20hours. After cooling to room temperature, the mixture was diluted with1.25 M HCl (60 ml) and washed with several portions of ether. The etherlayer was back-extracted with several portions of 1.25 M HCl. Thecombined aqueous layers were cooled in an ice bath and diluted withCHCl3 (30 ml). The mixture was stirred and solid K2CO3 was addedcarefully until pH 9. The phases were separated and the aqueous layerextracted with CHCl3 (3×30 ml) and the combined extracts washed withbrine and dried with Na2SO4. The solvent was evaporated leaving a darkbrown oil that was triturated with EtOAc. The liquid layer was decantedaway from the undesired solid residue. The residue was washed withseveral portions of EtOAc. The combined EtOAc (150 ml) was dried withNa2SO4 and the solvent evaporated leaving a homogeneous, brown oil (3.8g). This was purified via flash chromatography (90/10/1CH2Cl2/MeOH/NH4OH) affording a translucent, light-brown oil(crystallized upon long standing) (2.1 g, 60%). The benzyl6-bromohexanoate used above was synthesized from 6-bromohexanoic acid(1.0 eq.) (Aldrich Chemical Co.), benzyl alcohol (1.3 eq.), andp-toluenesulphonic acid (0.05 eq.) in refluxing cyclohexane (1.5 ml/mmol6-Br-hexanoic acid) with the aid of a Dean-Stark trap. After 2 hours,the solution was allowed to cool to room temperature and the solventremoved on a rotary evaporator. The residue was diluted with EtOAc,washed with 1 M NaHCO3, brine, dried with MgSO4 and the solventevaporated. The residue was distilled (benzyl alcohol forerun) affordinga clear, colorless liquid (87% yield) (bp 150-155∞ C., 4 mmHg).

Synthesis of Compound 3,[1R-(exo,exo)]-6-[[[3-(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]carbonyl]oxy]-hexanoicacid phenylmethyl ester, as illustrated in FIG. 1

Compound 3. Compound 2 (250 mg, 0.64 mmol) was dissolved in CH2Cl2 (1.5ml). The solution was cooled in an ice bath and then NEt3 (107 ul, 0.77mmol) and DMAP (4-dimethylamino-pyridine) (10 mg, 0.077 mmol) wereadded. The mixture was allowed to stir at room temperature for 6 hoursor until complete, as monitored by TLC. The mixture was diluted withEtOAc and washed with 3.5 M K2CO3, brine, dried with Na2SO4 and thesolvent evaporated. The residue was purified via flash chromatography(95/5/1 CH2Cl2/MeOH/NH40H) affording a thick, straw-colored oil (190 mg,60%).

Synthesis of Compound 4,[1R-(exo,exo)]-6-[[[3-(benzoyloxy)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]carbonyl]oxy]-hexanoicacid, as illustrated in FIG. 1

Compound 4. To a solution of compound 3 (136 mg, 0.275 mmol) in MeOH (3ml) was added 10% Pd/C (30 mg). The reaction mixture was shaken on aParr apparatus under 40 psi of hydrogen for 6 hours. After this time,the mixture was filtered through a pad of celite in a sintered glassfunnel washing with MeOH. The solvent was removed on a rotary evaporatorand the residue thoroughly dried under vacuum affording a colorless,hygroscopic solid that required no further purification (110 mg, 99%).

Synthesis of Compound 6 (FIG. 5; Step b)

Compound 6. Compound 2 (1.0 eq.) was dissolved in THF (1 ml/0.5 mmol 2)and the solution cooled in an ice bath. An ice-cold solution of LDA(lithium diisopropylamide) (1.0 eq.) (Aldrich) in THF was added. After 5minutes, a solution of freshly-prepared compound 8 (1.5 eq.) in THF (1ml/mmol 8) was added. The ice bath was removed and the mixture stirredat room temperature for 6 hours. After dilution with EtOAc and the usualworkup (see compound 3), the crude residue was purified via flashchromatography (95/5/1 EtOAc/MeOH/NH4OH) affording a thick,straw-colored oil (45% yield) as a 50/50 mixture of two diastereomersthat did not require separation for the following step.

Synthesis of Compound 7 (FIG. 5; Step c)

Compound 7. To a solution of compound 6 (136 mg, 0.275 mmol) in MeOH (3ml) was added 10% Pd/C (30 mg). The reaction mixture was shaken on aParr apparatus under 40 psi of hydrogen for 6 hours. After this time,the mixture was filtered through a pad of celite in a sintered glassfunnel washing with MeOH. The solvent was removed on a rotary evaporatorand the residue thoroughly dried under vacuum affording a colorless,hygroscopic solid that required no further purification (110 mg, 99%).The compound was obtained as a colorless, glassy solid (99% yield).

Synthesis of Compound 8 (FIG. 5; Steps a-b)

Compound 8. A solution of phenylphosphonic dichloride (1.0 eq.)(Aldrich) in CH2Cl2 (2 ml/mmol) was cooled in an ice bath. A solution ofbenzyl alcohol (2.0 eq.) and NEt3 (2.1 eq.) in CH2Cl2 (0.2 ml/mmol) wasadded dropwise. After stirring 16 hours at room temperature, the mixturewas diluted with EtOAc and washed with 1 M HCl, 1 M NaHCO3, brine, driedwith Na2SO4 and the solvent evaporated. The residue was purified viaflash chromatography (70/30 EtOAc/hexane) affording a white solid (84%yield). This material was converted to the phosphonyl chlorideimmediately before use as follows. To a solution of the dibenzyl ester(1.0 eq.) in CHCl3 (1 ml/mmol) was added PCl5 (1.0 eq.) (Aldrich). Themixture was heated to 45° C. in an oil bath. After 3 hours, the solutionwas allowed to cool to room temperature and then purged with SO2 gas(generated by heating NaHSO3) for several minutes. The solvent wasevaporated and the residue thoroughly dried under high vacuum at 45∞ C.for 30 minutes affording a thick, yellow oil (98% yield).

Synthesis of Compound 9 (FIG. 6)

Compound 9. Compound 9 is commercially available as (−) cocaine HCl(Sigma). The free-base was prepared from this material using a standardmethodology of expedient chloroform extraction from a basic (pH 9-10)(K2CO3) aqueous solution.

Synthesis of Compound 10 (FIG. 6; Step a)

Compound 10. To a solution of compound 9 (1.0 eq.) in benzene (2.5ml/mmol 9) was added a solution of trichloroethyl chloroformate (1.3eq.) (Aldrich) in benzene (0.5 ml/mmol). The solution was refluxed for18 hours. The mixture was cooled in an ice bath and formic acid (0.5eq.) was added and after 30 minutes NEt3 (0.3 eq.) was added. Afterstirring 1 hour, water (4 ml/mmol) was added and the mixture extractedthoroughly with ether. The ether extracts were washed with 6 M HCl,dried and evaporated affording the intermediate carbamate as a thick,colorless oil (80% yield). The oil was dissolved in DMF (1.5 ml/mmol)and the solution cooled in an ice bath. Formic acid (2.5 eq.) was addedand then activated zinc dust (3.5 eq.) was added in portions over 10minutes. After 15 minutes, the ice bath was removed and the mixturestirred at room temperature for 18 hours. The mixture was filtered ontocrushed ice washing with small portions of DMF. After the ice melted,the yellowish mixture was cooled in an ice bath and acidified withconcentrated HCl until pH 2. The cold mixture was extracted with etherand the aqueous layer cooled in an ice bath and made basic with NH4OH topH 9. The cold mixture was thoroughly extracted with ether, and thecombined extracts washed with brine, dried and evaporated affording athick, pale-yellow oil (41% yield) that required no furtherpurification.

Synthesis of Compound 11 (FIG. 6; Step b)

Compound 11. Compound 10 was dissolved in 1.25 M HCl (4 ml/mmol 10). Themixture was allowed to stir for 20 hours at 110-115° C. (gentle reflux).After cooling to room temperature, the mixture was thoroughly extractedwith ether to remove the benzoic acid by-product. The acidic aqueouslayer was evaporated on a rotary evaporator under high vacuum and thenthe residue was redissolved in water and lyophillized affording a white,very hygroscopic solid that required no further purification (98%yield).

Synthesis of Compound 12 (FIG. 6; Step c)

Compound 12. Compound 11 was dissolved in MeOH (5 ml/mmol 11) and thesolution thoroughly purged with HCl gas. The reaction flask was sealedand the solution stirred for 18 hours at room temperature and thenrefluxed for 5 hours. The solvent was evaporated and the hygroscopic HClsalt was converted to the free base (see compound 9) affording a clear,straw-colored oil (78% yield) that crystallized upon prolonged standing.

Synthesis of Compound 13 (FIG. 6; Step d)

Compound 13. Compound 12 (1.0 eq.) was dissolved in CH3CN (4 ml/mmol12). NEt3 (1.2 eq.), benzyl 4-bromobutanoate (1.2 eq.) (prepared in asimilar fashion to the hexanoate, see compound 2) andtetrabutyl-ammoniumiodide (0.1 eq.) were then added. The mixture washeated at 50° C. for 24 hours. After dilution with EtOAc and the usualworkup (see compound 3), the crude residue was purified via flashchromatography (95/5/1 EtOAc/MeOH/NH4OH) affording a clear,straw-colored oil (58% yield).

Synthesis of Compound 14 (FIG. 6; Step e)

Compound 14. Compound 13 (1.0 eq.) was dissolved in THF (1 ml/0.5 mmol2) and the solution cooled in an ice bath. An ice-cold solution of LDA(lithium diisopropylamide) (1.0 eq.) (Aldrich) in THF was added. After 5minutes, a solution of freshly-prepared compound 8 (1.5 eq.) in THF (1ml/mmol 8) was added. The ice bath was removed and the mixture stirredat room temperature for 6 hours. After dilution with EtOAc and the usualworkup (see compound 3), the crude residue was purified via flashchromatography (95/5/1 EtOAc/MeOH/NH4OH) affording a thick,straw-colored oil (45% yield) as a 50/50 mixture of two diastereomersthat did not require separation for the following step. The compound wasobtained after flash chromatography (95/5 EtOAc/MeOH) as a clear,straw-colored oil composed of a 50/50 mixture of two diastereomers (40%yield).

Synthesis of Compound 15 (FIG. 6; Step f)

Compound 15. To a solution of compound 14 (136 mg, 0.275 mmol) in MeOH(3 ml) was added 10% Pd/C (30 mg). The reaction mixture was shaken on aParr apparatus under 40 psi of hydrogen for 6 hours. After this time,the mixture was filtered through a pad of celite in a sintered glassfunnel washing with MeOH. The solvent was removed on a rotary evaporatorand the residue thoroughly dried under vacuum affording a colorless,hygroscopic solid that required no further purification (110 mg, 99%).The compound was obtained as colorless, hygroscopic crystals (99%yield).

Synthesis of Compound 16 (FIG. 7; Step a)

Compound 16. Compound 1 was dissolved in MeOH (5 ml/mmol 11) and thesolution thoroughly purged with HCl gas. The reaction flask was sealedand the solution stirred for 18 hours at room temperature and thenrefluxed for 5 hours. The solvent was evaporated and the hygroscopic HClsalt was converted to the free base (see compound 9) affording a clear,straw-colored oil (78% yield) that crystallized upon prolonged standing.The compound was obtained as a clear, colorless oil (78% yield) thatcrystallized upon prolonged standing.

Synthesis of Compound 17 (FIG. 7; Step b)

Compound 17. Compound 16 (1.0 eq.) was dissolved in THF (1 ml/0.5 mmol2) and the solution cooled in an ice bath. An ice-cold solution of LDA(lithium diisopropylamide) (1.0 eq.) (Aldrich) in THF was added. After 5minutes, a solution of freshly-prepared compound 8 (1.5 eq.) in THF (1ml/mmol 8) was added. The ice bath was removed and the mixture stirredat room temperature for 6 hours. After dilution with EtOAc and the usualworkup (see compound 3), the crude residue was purified via flashchromatography (95/5/1 EtOAc/MeOH/NH4OH) affording a thick,straw-colored oil (45% yield) as a 50/50 mixture of two diastereomersthat did not require separation for the following step. The compound wasobtained after flash chromatography (95/5/1 EtOAc/MeOH/NH4OH) as apale-yellow oil composed of a 50/50 mixture of two diastereomers (31%yield).

Synthesis of Compound 18 (FIG. 7; Step c)

Compound 18. To a solution of compound 17 (136 mg, 0.275 mmol) in MeOH(3 ml) was added 10% Pd/C (30 mg). The reaction mixture was shaken on aParr apparatus under 40 psi of hydrogen for 6 hours. After this time,the mixture was filtered through a pad of celite in a sintered glassfunnel washing with MeOH. The solvent was removed on a rotary evaporatorand the residue thoroughly dried under vacuum affording a colorless,hygroscopic solid that required no further purification (110 mg, 99%).The compound was obtained as colorless, hygroscopic crystals (99%yield).

Synthesis of Compound 19 (FIG. 7; Steps a-e)

Compound 19. Step a: A water-jacketed photochemical reactor (250 mlcapacity) (Ace Glass Co.) was charged with a stirring bar, 4-iodophenol(71 g, 0.322 mol) (Aldrich) and trimethyl phosphite (200 g, 1.61 mol)(Aldrich). The solution was purged with nitrogen and cooled with 50/50water/ethylene glycol (−2° C.) using a circulating bath (Lauda). Awater-jacketed high-pressure mercury lamp (Hanovia) was put in place andthe entire apparatus wrapped with aluminum foil. The solution wasirradiated for 24 hours under nitrogen. The excess trimethyl phosphitewas then removed via distillation leaving a residue that contained 60%of the desired product and 40% dimethyl methylphosphonate. The latterwas removed via distillation under high vacuum leaving a thick syrup (80g). This material was purified in portions via flash chromatography(95/5 EtOAc/MeOH) affording a thick, pale-yellow oil (52 g, 80%). Stepb: The compound from step a (1.0 eq.) was dissolved in DMF (0.5 ml/mmol)and added dropwise to a stirring, ice-cold suspension of oil-free NaH(1.1 eq.) in DMF (1 ml/mmol). After 15 minutes, benzyl 4-bromobutanoate(1.1 eq.) (see compound 13) was added followed bytetrabutylammoniumiodide (0.011 eq.). The ice bath was removed and themixture stirred for 2 hours at room temperature. EtOAc was added and themixture washed thoroughly with brine, dried with Na2SO4, and solventevaporated. The residue was purified via flash chromatography (EtOAc)affording a pale-yellow liquid (69% yield). Step c: The compound fromstep b (1.0 eq.) was dissolved in CH2Cl2 (1 ml/mmol) and thentrimethylsilylbromide (2.2 eq.) was slowly added. The solution wasstirred at room temperature for 2 hours. The solvent mixture wasevaporated under reduced pressure and the residue redissolved in CH2Cl2(1 ml/mmol) containing a drop of DMF and then oxalyl chloride was slowlyadded. The solution was stirred at room temperature for 1 hour. Thesolvent mixture was evaporated under reduced pressure and the residueredissolved in CH2Cl2 (2 ml/mmol) and cooled in an ice bath. A solutionof benzyl alcohol (2.1 eq.) and NEt3 (2.2 eq.) in CH2Cl2 (0.2 ml/mmol)was then added. The ice bath was removed and the mixture stirred for 18hours at room temperature. After dilution with EtOAc, the mixture waswashed with 1 M HCl, 1 NaHCO3, brine, dried with Na2SO4 and the solventevaporated. The residue was purified via flash chromatography (50/50EtOAc/hexane) affording a clear, pale-yellow liquid (60% yield). Step d:The compound from step c was used to prepare compound 19 (99% yield)(see procedure for compound 8).

Synthesis of Compound 20 (FIG. 8; Step b)

Compound 20. Compound 2 (1.0 eq.) was dissolved in THF (1 ml/0.5 mmol 2)and the solution cooled in an ice bath. An ice-cold solution of LDA(lithium diisopropylamide) (1.0 eq.) (Aldrich) in THF was added. After 5minutes, a solution of freshly-prepared compound 8 (1.5 eq.) in THF (1ml/mmol 8) was added. The ice bath was removed and the mixture stirredat room temperature for 6 hours. After dilution with EtOAc and the usualworkup (see compound 3), the crude residue was purified via flashchromatography (95/5/1 EtOAc/MeOH/NH4OH) affording a thick,straw-colored oil (45% yield) as a 50/50 mixture of two diastereomersthat did not require separation for the following step. Step a: Asolution of dibenzylphosphite (1.0 eq.) (Aldrich) in DMF (1 ml/mmol) wasadded dropwise to a stirring, ice-cold suspension of oil-free NaH (1.1eq.) in DMF (1 ml/mmol). After 15 minutes, bromohexane (1.1 eq.)(Aldrich) was added followed by tetrabutylammonium-iodide (0.011 eq.).The ice bath was removed and the mixture stirred for 2 hours at roomtemperature. EtOAc was added and the mixture washed thoroughly withbrine, dried with Na2SO4, and the solvent evaporated. The residue waspurified via flash chromatography (80/20 CH2Cl2/EtOAc) affording a clearoil (52% yield). Step b: The compound from step a was used to preparecompound 22 (99% yield) (see procedure for compound 8). The compound wasobtained after flash chromatography (90/10 EtOAc/MeOH) as a clear,straw-colored oil composed of a 50/50 mixture of two diastereomers (30%yield).

Synthesis of Compound 21 (FIG. 8; Step c)

Compound 21. To a solution of compound 20 (136 mg, 0.275 mmol) in MeOH(3 ml) was added 10% Pd/C (30 mg). The reaction mixture was shaken on aParr apparatus under 40 psi of hydrogen for 6 hours. After this time,the mixture was filtered through a pad of celite in a sintered glassfunnel washing with MeOH. The solvent was removed on a rotary evaporatorand the residue thoroughly dried under vacuum affording a colorless,hygroscopic solid that required no further purification (110 mg, 99%).The compound was obtained as colorless, hygroscopic crystals (99%yield).

Synthesis of Compound 22 (FIG. 8; Steps a-b)

Compound 22. Step a: A solution of dibenzylphosphite (1.0 eq.) (Aldrich)in DMF (1 ml/mmol) was added dropwise to a stirring, ice-cold suspensionof oil-free NaH (1.1 eq.) in DMF (1 ml/mmol). After 15 minutes,bromohexane (1.1 eq.) (Aldrich) was added followed bytetrabutylammonium-iodide (0.011 eq.). The ice bath was removed and themixture stirred for 2 hours at room temperature. EtOAc was added and themixture washed thoroughly with brine, dried with Na2SO4, and the solventevaporated. The residue was purified via flash chromatography (80/20CH2Cl2/EtOAc) affording a clear oil (52% yield). Step b: The compoundfrom step a was used to prepare compound 22 (99% yield) (see procedurefor compound 8).

Synthesis of Compound 23 (FIG. 9; Step b)

Compound 23. Compound 2 (1.0 eq.) was dissolved in THF (1 ml/0.5 mmol 2)and the solution cooled in an ice bath. An ice-cold solution of LDA(lithium diisopropylamide) (1.0 eq.) (Aldrich) in THF was added. After 5minutes, a solution of freshly-prepared compound 8 (1.5 eq.) in THF (1ml/mmol 8) was added. The ice bath was removed and the mixture stirredat room temperature for 6 hours. After dilution with EtOAc and the usualworkup (see compound 3), the crude residue was purified via flashchromatography (95/5/1 EtOAc/MeOH/NH4OH) affording a thick,straw-colored oil (45% yield) as a 50/50 mixture of two diastereomersthat did not require separation for the following step. The compound wasobtained after flash chromatography (90/10 EtOAc/MeOH) as a clear,straw-colored oil composed of 50/50 mixture of two diastereomers (34%yield). A solution of phenylphosphonic dichloride (1.0 eq.) (Aldrich) inCH2Cl2 (2 ml/mmol) was cooled in an ice bath. A solution oftrifluoroethanol (2.2 eq.) and NEt3 (2.5 eq.) in CH2Cl2 (0.2 ml/mmol)was added dropwise. After stirring 6 hours at room temperature, themixture was diluted with EtOAc and washed with 1 M HCl, 1 M NaHCO3,brine, dried with Na2SO4 and the solvent evaporated. The residue waspurified via flash chromatography (CH2Cl2) affording a colorless oil(88% yield).

Synthesis of Compound 24 (FIG. 9, Step c)

Compound 24. To a solution of compound 23 (136 mg, 0.275 mmol) in MeOH(3 ml) was added 10% Pd/C (30 mg). The reaction mixture was shaken on aParr apparatus under 40 psi of hydrogen for 6 hours. After this time,the mixture was filtered through a pad of celite in a sintered glassfunnel washing with MeOH. The solvent was removed on a rotary evaporatorand the residue thoroughly dried under vacuum affording a colorless,hygroscopic solid that required no further purification (110 mg, 99%).The compound was obtained as colorless, hygroscopic crystals (99%yield).

Synthesis of Compound 25 (FIG. 9, Step a)

Compound 25. A solution of phenylphosphonic dichloride (1.0 eq.)(Aldrich) in CH2Cl2 (2 ml/mmol) was cooled in an ice bath. A solution oftrifluoroethanol (2.2 eq.) and NEt3 (2.5 eq.) in CH2Cl2 (0.2 ml/mmol)was added dropwise. After stirring 6 hours at room temperature, themixture was diluted with EtOAc and washed with 1 M HCl, 1 M NaHCO3,brine, dried with Na2SO4 and the solvent evaporated. The residue waspurified via flash chromatography (CH2Cl2) affording a colorless oil(88% yield).

Synthesis of Compound 26 (FIG. 10, Step a)

Compound 26. Compound 1 (2.0 g, 9.0 mmol) was suspended in a mixture ofpyridine (250 ml, 3.1 mmol), finely powdered NaOH (760 mg, 19 mmol) andbenzylbromide (7.8 g, 27 mmol). The mixture was heated to 80° C. andallowed to stir for 20 hours. After cooling to room temperature, themixture was diluted with 1.25 M HCl (60 ml) and washed with severalportions of ether. The ether layer was back-extracted with severalportions of 1.25 M HCl. The combined aqueous layers were cooled in anice bath and diluted with CHCl3 (30 ml). The mixture was stirred andsolid K2CO3 was added carefully until pH 9. The phases were separatedand the aqueous layer extracted with CHCl3 (3×30 ml) and the combinedextracts washed with brine and dried with Na2SO4. The solvent wasevaporated leaving a dark brown oil that was triturated with EtOAc. Theliquid layer was decanted away from the undesired solid residue. Theresidue was washed with several portions of EtOAc. The combined EtOAc(150 ml) was dried with Na2SO4 and the solvent evaporated leaving ahomogeneous, brown oil (3.8 g). This was purified via flashchromatography (90/10/1 CH2Cl2/MeOH/NH4OH) affording a translucent,light-brown oil (crystallized upon long standing) (2.1 g, 60%). Thebenzyl 6-bromohexanoate used above was synthesized from 6-bromohexanoicacid (1.0 eq.) (Aldrich Chemical Co.), benzyl alcohol (1.3 eq.), andp-toluenesulphonic acid (0.05 eq.) in refluxing cyclohexane (1.5 ml/mmol6-Br-hexanoic acid) with the aid of a Dean-Stark trap. After 2 hours,the solution was allowed to cool to room temperature and the solventremoved on a rotary evaporator. The residue was diluted with EtOAc,washed with 1 M NaHCO3, brine, dried with MgSO4 and the solventevaporated. The residue was distilled (benzyl alcohol forerun) affordinga clear, colorless liquid (87% yield) (bp 150-155° C., 4 mmHg).

Synthesis of Compound 27 (FIG. 10, Step b)

Compound 27. Compound 26 (250 mg, 0.64 mmol) was dissolved in CH2Cl2(1.5 ml). The solution was cooled in an ice bath and then NEt3 (107 ul,0.77 mmol), compound 31 (1.1 equivalents; vida infra) and DMAP(4-dimethylamino-pyridine) (10 mg, 0.077 mmol) were added. The mixturewas allowed to stir at room temperature for 6 hours or until complete,as monitored by TLC. The mixture was diluted with EtOAc and washed with3.5 M K2CO3, brine, dried with Na2SO4 and the solvent evaporated. Theresidue was purified via flash chromatography (95/5/1 CH2Cl2/MeOH/NH4OH)affording a thick, straw-colored oil (190 mg, 60%).

Synthesis of Compound 28 (FIG. 10, Step c)

Compound 28. To a solution of compound 27 (136 mg, 0.275 mmol) in MeOH(3 ml) was added 10% Pd/C (30 mg). The reaction mixture was shaken on aParr apparatus under 40 psi of hydrogen for 6 hours. After this time,the mixture was filtered through a pad of celite in a sintered glassfunnel washing with MeOH. The solvent was removed on a rotary evaporatorand the residue thoroughly dried under vacuum affording a colorless,hygroscopic solid that required no further purification (110 mg, 99%).

Synthesis of Compound 29 (FIG. 10, Step d)

Compound 29. The compound 28 (1.0 eq.), methylamine hydrochloride (1.2eq.) and NEt3 (2.2 eq.) are dissolved in DMF (0.5 M solution), and then1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) (1.3eq.) and DMAP (0.013 eq.) is added at 0° C. The reaction mixture isstirred for 24 hours at room temperature. The mixture is diluted withEtOAc and the organic layer washed with saturated 1 M HCl, 1 M NaHCO3,and brine. After drying over Na2SO4 and removal of the solvent, theresidue is purified via flash chromatography.

Synthesis of Compound 30 (FIG. 10, Step e)

Compound 30. The compound 29 is dissolved in CH2Cl2 (1.0 M solution) andcooled in an ice bath. Trifluoroacetic acid (3.0 eq.) is added and thesolution stirred for 1 hour at room temperature. The solvent andvolatiles are then thoroughly evaporated. The compound is obtained as asalt and does not require further purification.

Synthesis of Compound 31 (FIG. 10, Steps a-c)

Compound 31. Step a: Methyl-p-hydroxybenzoate (1.0 eq. (Aldrich) isdissolved in DMF (1.0 M solution) and added dropwise to a stirring,ice-cold suspension of oil-free NaH (1.1 eq.) in DMF (0.5 M suspension).After 15 minutes, benzyl 4-bromobutanoate (1.1 eq.) (see compound 13) isadded followed by tetrabutylammoniumiodide (0.011 eq.). The ice bath isremoved and the mixture stirred for 2 hours at room temperature. EtOAcis added and the mixture washed thoroughly with brine, dried withNa2SO4, and solvent evaporated. The residue is purified via flashchromatography. Step b: The compound from step a is dissolved in MeOH(0.5 M solution) and cooled in an ice bath. A solution of LiOH (20 eq.)in water (0.5 M solution) is then added. After 3 hours, the mixture isacidified, the compound extracted with EtOAc and the organic layerwashed with brine and dried. Step c: The compound from step b isdissolved in THF (1.0 M solution) containing a drop of DMF and thesolution cooled in an ice bath. Oxalyl chloride (1.1 eq.) is added andthe solution stirred for 1 hour at room temperature. The solvent andvolatiles are then thoroughly evaporated and the compound used withoutfurther purification. The t-butyl 4-bromobutanoate used in step a issynthesized from 4-bromobutanoic acid (1.0 eq.) (Aldrich) suspended inhexane containing a catalytic amount of Amberlyst-H+ resin and excessisobutylene. The mixture is stirred for 16 hours at room temperature.After filtration and evaporation of the solvent, the compound is usedwithout further purification.

Synthesis of Compound 32 (FIG. 11, Step a)

Compound 32. The compound 11 (1.0 eq.) is suspended in CH2Cl2 containinga catalytic amount of sulfuric acid and excess isobutylene. The mixtureis stirred for 16 hours at room temperature. After evaporation of thesolvent, the residue is partitioned between chloroform and K2CO3solution. The organic layer is dried and evaporated to afford thecompound as the free base that is used without further purification.

Synthesis of Compound 33 (FIG. 11, Step b)

Compound 33. Compound 32 (1.0 eq.) was dissolved in CH3CN (4 ml/mmol12). NEt3 (1.2 eq.), benzyl 4-bromobutanoate (1.2 eq.) (prepared in asimilar fashion to the hexanoate, see compound 2) andtetrabutyl-ammoniumiodide (0.1 eq.) were then added. The mixture washeated at 50° C. for 24 hours. After dilution with EtOAc and the usualworkup (see compound 3), the crude residue was purified via flashchromatography (95/5/1 EtOAc/MeOH/NH4OH) affording a clear,straw-colored oil (58% yield).

Synthesis of Compound 34 (FIG. 11, Step c)

Compound 34. Compound 33 (250 mg, 0.64 mmol) was dissolved in CH2Cl2(1.5 ml). The solution was cooled in an ice bath and then NEt3 (107 ul,0.77 mmol) and DMAP (4-dimethylamino-pyridine) (10 mg, 0.077 mmol) wereadded. The mixture was allowed to stir at room temperature for 6 hoursor until complete, as monitored by TLC. The mixture was diluted withEtOAc and washed with 3.5 M K2CO3, brine, dried with Na2SO4 and thesolvent evaporated. The residue was purified via flash chromatography(95/5/1 CH2Cl2/MeOH/NH4OH) affording a thick, straw-colored oil (190 mg,60%).

Synthesis of Compound 35 (FIG. 11, Step d)

Compound 35. The compound 34 is dissolved in CH2Cl2 (1.0 M solution) andcooled in an ice bath. Trifluoroacetic acid (3.0 eq.) is added and thesolution stirred for 1 hour at room temperature. The solvent andvolatiles are then thoroughly evaporated. The compound is obtained as asalt and does not require further purification.

Synthesis of Compound 36 (FIG. 11, Step e)

Compound 36. The compound 35 (1.0 eq.), methylamine hydrochloride (1.2eq.) and NEt3 (2.2 eq.) are dissolved in DMF (0.5 M solution), and then1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) (1.3eq.) and DMAP (0.013 eq.) is added at 0° C. The reaction mixture isstirred for 24 hours at room temperature. The mixture is diluted withEtOAc and the organic layer washed with saturated 1 M HCl, 1 M NaHCO3,and brine. After drying over Na2SO4 and removal of the solvent, theresidue is purified via flash chromatography.

Synthesis of Compound 37 (FIG. 11, Step f)

Compound 37. To a solution of compound 35 (136 mg, 0.275 mmol) in MeOH(3 ml) was added 10% Pd/C (30 mg). The reaction mixture was shaken on aParr apparatus under 40 psi of hydrogen for 6 hours. After this time,the mixture was filtered through a pad of celite in a sintered glassfunnel washing with MeOH. The solvent was removed on a rotary evaporatorand the residue thoroughly dried under vacuum affording a colorless,hygroscopic solid that required no further purification (110 mg, 99%).

Synthesis of Compound 39,(1R)-3-amino-8-methyl-8-azabicyclo[3.2.1]oct-2-ene-2-carboxylic acidmethyl ester (FIG. 12; Step a)

Compound 39. Ammonium acetate (391 mg, 5.07 mmol) was added to asolution of (1R)-(+)-2-carbomethoxy-3-tropinone 38 (200 mg, 1.01 mmol)(S. P. Findlay, J. Org. Chem. (1957): vol: 22, p 1385-1394; F. I.Carroll, et al., J. Org. Chem. (1982): vol. 47, p 13-19; A. H. Lewin, etal., J. Heterocyclic Chem. (1987): vol. 24, p 19-21; P. C. Meltzer, etal., J. Med. Chem. (1994): vol. 37, p 2001-2010) in benzene/acetic acid(17.5 ml/0.1 ml). The mixture was refluxed for 10 hours. To thisreaction mixture was added CH2Cl2 and c-NH4OH. The organic layer wascollected and dried over Na2SO4. After removal of the solvent, theresidue was purified via flash chromatography (CH2Cl2/MeOH/c-NH4OH;20/1/0.02 to 5/1/0.02) affording colorless crystals (160 mg, 80%). 1HNMR (300 MHz, CDCl3): d 1.42-1.53 (1H, m), 1.68-1.81 (3H, m), 2.01-2.19(3H, m), 2.31 (3H, s), 2.73 (1H, dd, J=4.9, 17.1 Hz), 3.36 (1H, t, J=5.7Hz), 3.67 (3H, 5), 3.82 (1H, d, J=5.1 Hz). 13C NMR (75 MHz, CDCl3): d29.0, 33.9, 34.9, 36.6, 50.4, 57.4, 57.9, 150.5, 153.8, 169.2. mp 95-98°C. HRMS (FAB) m/e calcd for C10H16N2O2+H+, 197.1290; found 197.1287.

Synthesis of Compound 40a,[1R-(2-endo,3-exo)]-3-(benzoylamino)-8-methyl-8-azabicyclo[3.2.1]-octane-2-carboxylicacid methyl ester; compound 40b,[1R-(endo,endo)]-3-(benzoylamino)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylicacid methyl ester; Compound 40c,[1R-(2-exo,3-endo)]-3-(benzoylamino)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylicacid methyl ester (FIG. 12; Step b)

Compounds 40a, 40b, 40c. Compound 39 (667 mg, 3.40 mmol) was dissolvedin MeOH (6 ml), then 2 M HCl in dioxane/MeOH was added and the pHadjusted to pH 4. Sodium cyanoborohydride (214 mg, 3.40 mmol) was addedand the mixture stirred for 18 hours at room temperature. The solventwas removed in vacuo and the residue was dissolved in dioxane/H2O (2ml/2 ml). To this solution was added NaHCO3 (1.43 g, 17.0 mmol) andbenzoyl chloride (0.79 ml, 6.80 mmol), and the mixture stirred for 20hours. The mixture was diluted with CH2Cl2 and c-NH4OH and the organiclayer collected and dried over Na2SO4. After removal of the solvent, theresidue was purified via flash chromatography (CH2Cl2/MeOH/c-NH4OH;20/1/0.02 to 5/1/0.02) affording, in order of polarity: 40a (195 mg,19%), 40b (196 mg, 19%) and 40c (62 mg, 6%) as oils. Compound 40a: 1HNMR (300 MHz, CD3OD): d 1.58-2.31 (8H, m), 2.45 (3H, s), 3.06 (1H, dd,J=2.7, 11.5 Hz), 3.28-3.32 (1H, m), 3.45-3.50 (1H, m), 3.67 (3H, s),4.58 (1H, dt, J=6.2, 11.4 Hz), 7.48-7.60 (3H, m), 7.78-7.84 (2H, m). 13CNMR (75 MHz, CDCl3): d 23.6, 26.2, 35.6, 38.5, 43.2, 49.6, 51.9, 60.4,62.8, 126.9, 128.4, 131.3, 134.5, 166.8, 172.5. HRMS (FAB) m/e calcd forC17H22N2O3+H+, 303.1709; found 303.1724. Compound 40b: 1H NMR (300 MHz,CD3OD): d 1.92-2.43 (11H, m) including 2.40 (3H, s), 3.24-3.31 (2H, m),3.41-3.48 (1H, m), 3.66 (3H, s), 4.61 (1H, t, J=6.2 Hz), 7.42-7.62 (3H,m), 7.73-7.78 (2H, m). 13C NMR (75 MHz, CDCl3): d 23.8, 25.2, 36.0,39.9, 43.3, 47.3, 51.8, 59.9, 61.7, 126.7, 128.7, 131.5, 134.6, 166.7,173.4. HRMS (FAB) m/e calcd for C17H22N2O3+H+, 303.1709; found 303.1720.Compound 40c: 1H NMR (300 MHz, CD3OD): d 1.80-1.89 (1H, m), 1.95-2.40(8H, m) including 2.35 (3H, s), 2.93-2.98 (1H, m), 2.95-2.99 (1H, m),3.20-3.28 (1H, m), 3.66-3.71 (1H, m) 3.78 (3H, s), 4.55 (1H, dt, J 2.2,7.2 Hz), 7.48-7.62 (3H, m), 7.75-7.82 (2H, m). 13C NMR (75 MHz, CDCl3):d 24.5, 25.6, 36.2, 41.6, 42.7, 51.7, 52.1, 60.9, 63.2, 126.6, 128.7,131.5, 134.5, 166.3, 172.8. HRMS (FAB) m/e calcd for C17H22N2O3+H+,3.03.1709; found 303.1704.

Synthesis of Compound 41a,[1R-(exo,exo)]-3-(benzoylamino)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylicacid; Compound 41b,[1R-(2-endo,3-exo)]-3-(benzoylamino)-8-methyl-8-azabicyclo-[3.2.1]octane-2-carboxylicacid (FIG. 12; Step c)

Compounds 41a, 41b. Compound 40a (138 mg, 0.456 mmol) was dissolved inwater (10 ml) and the solution refluxed for 24 hours. The solution waslyophilized affording a 50/50 mixture of diastereomers as a fluffypowder (123 mg, 93%). 1H NMR (300 MHz, CD3OD): d 1.98-2.52 (6H, m), 2.45and 2.88 (3H, each s), 2.94 (0.4H, dd, J 2.7, 6.5 Hz), 3.18 (0.6H, dd,J=2.5, 11.5 Hz), 3.88-3.95 (1H, m), 4.05-4.10 (1H, m), 4.42-4.62 (1H,m), 7.47-7.60 (3H, m), 7.79-7.88 (2H, m). 13C NMR (125 MHz, D2O): d20.5, 22.5, 23.0, 30.2, 31.9, 34.1, 37.3, 37.8, 39.9, 41.9, 48.2, 51.6,62.0, 63.1, 64.4, 65.0, 126.5, 128.1, 131.5, 131.6, 132.8, 132.9, 169.9,170.0, 174.2, 176.8. HRMS (FAB) m/e calcd for C16H20N2O3+Na+, 311.1372;found 311.1382.

Synthesis of Compound 42a,[1R-(exo,exo)]-6-[[[3-(benzoylamino)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]carbonyl]amino]-hexanoicacid phenylmethyl ester; Compound 42b,[1R-(2-endo,3-exo)]-6-[[[3-(benzoylamino)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]carbonyl]-amino]-hexanoicacid phenylmethyl ester (FIG. 12; Step d)

Compounds 42a, 42b. The mixture of 41a and 41b (39 mg, 0.14 mmol),6-aminohexanoic acid benzyl ester tosylate (64 mg, 0.16 mmol) and NEt3(23 ml, 0.16 mmol) were dissolved in CH2Cl2 (0.7 ml) and then EDC (33.6mg, 0.18 mmol) and DMAP (1.6 mg, 0.014 mmol) were added at 0° C. Thereaction mixture was stirred for 24 hours at room temperature. Themixture was diluted with CH2Cl2 and the organic layer washed withsaturated NaHCO3 and water. After drying over Na2SO4 and removal of thesolvent, the residue was purified by preparative TLC(CH2Cl2/MeOH/c-NH4OH; 5/1/0.02) affording 42a (12 mg, 18%) and 42b (27mg, 41%) as oils. Compound 42a: 1H NMR (300 MHz, CD3OD): d 1.34-2.45(17H, m) including 2.32 (3H, s), 2.78 (1H, dd, J=2.7, 6.4 Hz), 3.17-3.35(3H, m), 3.41-3.48 (1H, m), 4.45 (1H, dt, J=6.3, 12.5 Hz), 5.12 (2H, s),7.35-7.51 (8H, m), 7.75-7.82 (2H, m). 13C NMR (125 MHz, CDCl3): d 24.5,24.7, 25.6, 26.6,. 29.3, 34.1, 36.3, 38.8, 40.6, 41.5, 49.7, 60.9, 63.2,66.1, 127.0, 128.2 (overlapping, 128.22), 128.5, 128.6, 131.3, 134.2,136.0, 166.7, 172.9, 173.3. HRMS (FAB) m/e calcd for C29H37N3O4+H+,492.2862; found 492.2873. Compound 42b: 1H NMR (300 MHz, CD3OD): d1.18-1.58 (6H, m), 2.08-2.68 (8H, m), 2.81 (3H, s), 3.02-3.26 (2H, m),3.28 (1H, dt, J=2.5, 11.3 Hz), 3.85-3.95 (2H, m), 4.62-4.74 (1H, m),5.12 (2H, s), 7.35-7.55 (8H, m), 7.81-7.90 (2H, m). 13C NMR (125 MHz,CDCl3): d 23.5, 24.4, 26.1, 26.3, 28.9, 33.9, 36.6, 38.6, 39.3, 42.0,52.6, 60.8, 64.1, 66.0, 126.9, 128.1, 128.4, 128.5, 131.8, 133.4, 135.9,167.6, 170.8, 173.2. HRMS (FAB) m/e calcd for C29H37N3O4+Cs+, 624.1838;found 624.1820.

Synthesis of Compound 43,[1R-(exo,exo)]-6-[[[3-(benzoylamino)-8-methyl-8-azabicyclo[3.2.1]oct-2-yl]carbonyl]amino]-hexanoicacid (FIG. 12; Step e)

Compound 43. Compound 42a (17 mg, 0.0346 mmol) was dissolved in MeOH (1ml) and then 10% Pd/C (20 mg) was added. The reaction mixture wasstirred for 1 hour under H2 (1 atm) at room temperature then filteredand concentrated. The residue was dissolved in water and lyophilizedaffording a fluffy powder (13 mg, 94%). 1H NMR (300 MHz, CD3OD): d1.20-1.58 (6H, m), 2.03-2.27 (5H, m), 2.38-2.67 (m, 3H), 2.80 (3H, s),3.13 (1H, dd, J=2.6, 6.3 Hz), 3.15-3.33 (2H, m), 3.92-3.97 (1H, m),4.05-4.10 (1H, m), 4.60 (1H, dt, J=6.4, 12.8 Hz), 7.48-7.61 (3H, m),7.81-7.90 (2H, m). 13C NMR (75 MHz, D2O): d 22.0, 22.9, 24.5, 25.2,27.4, 31.7, 36.4, 37.7, 38.5, 40.7, 45.4, 62.4, 64.0, 126.5, 128.1,131.9, 132.1, 170.3, 171.9, 182.9. [a]/O(D,25)=−75.60 (c=0.5, methanol).HRMS (FAB) m/e calcd for C22H31N3O4+Na+, 402.2393; found 402.2405.

Synthesis of Compound 44 (Scheme 8)

Compound 44. Compound 43 was suspended in 0.10 Molar DMF at 0° C. Next,1.1 equivalents sufosuccinimide (Aldrich) and 1.1 equivalents EDC(1-3-Dimethylaminopropyl)-3-ethyl-carbo-diimide-hydrochloride Aldrich)were added and the mixture was stirred for 2 hours at 25° C. Next,either 1.1 equivalents KLH (keyhole limpet hemacyanin; Sigma) or 1.1equivalents BSA (bovine serum albumin; Sigma) was added and the mixturewas stirred at 25° C. for 12 hours. The mixture was next quenched withsuccessive saturated solution washes of ammonium chloride, water anddried over magnesium sulfate. The compound was purified via reversephase HPLC to afford compound 44.

What is claimed is:
 1. A compound having a formula selected from thegroup consisting of:

wherein n and m are integers from 2 to
 8. 2. The compound of claim 1,wherein said compound has the following formula:

wherein n is an integer from 2 to
 8. 3. The compound of claim 2, whereinn is an integer from 4 to
 6. 4. The compound of claim 3, wherein n is 5.5. A compound having a formula selected from the group consisting of:

wherein n and m are integers from 2 to
 8. 6. The compound of claim 5,wherein said compound has the following formula:

wherein n is an integer from 2 to
 8. 7. The compound of claim 6, whereinn is an integer from 4 to
 6. 8. The compound of claim 7, wherein is 5.9. Antisera resulting from immunization with a compound having a formulaselected from the group consisting of:

wherein n and m integers from 2 to
 8. 10. The antisera of claim 9,wherein said compound has the following formula:

wherein n is an integer from 2 to
 8. 11. The antisera of claim 10,wherein in said compound n is an integer from 4 to
 6. 12. The antiseraof claim 11, wherein in said compound n is
 5. 13. A monoclonal antibodyresulting from immunization with a compound having a formula selectedfrom the group consisting of:

wherein n and m are integers from 2 to 8, isolation of an anti-cocaineantibody producing cell which expresses said monoclonal antibody,cloning of said cell, and isolation of said monoclonal antibodyexpressed by said cloned cell.
 14. The monoclonal antibody of claim 13,wherein said compound has the following formula:

wherein n is an integer from 2 to
 8. 15. The monoclonal antibody ofclaim 14, wherein in said compound n is an integer from 4 to
 6. 16. Themonoclonal antibody of claim 15, wherein in said compound n is
 5. 17. Amethod of producing antisera specific for cocaine, said methodcomprising: immunizing a subject with a compound having a formulaselected from the group consisting of:

wherein n and m are integers from 2 to 8; and isolating antisera fromsaid subject.
 18. The method of claim 17, wherein said compound has thefollowing formula:

wherein n is an integer from 2 to
 8. 19. The method of claim 18, whereinin said compound n is an integer from 4 to
 6. 20. The method of claim19, wherein in said compound n is
 5. 21. A method of producing amonoclonal antibody specific for cocaine, said method comprising:immunizing a subject with a compound having a formula selected from thegroup consisting of:

wherein n and m are integers from 2 to 8; isolating an anti-cocaineantibody producing cell from said subject which expresses saidmonoclonal antibody and cloning said cell; and isolating said monoclonalantibody produced by said cloned cell.
 22. The method of claim 21,wherein said compound has the following formula:

wherein n is an integer from 2 to
 8. 23. The method of claim 22, whereinin said compound n is an integer from 4 to
 6. 24. The method of claim23, wherein in said compound n is
 5. 25. A method of suppressingpsychoactive effects of cocaine within a subject, said methodcomprising: administering an anti-cocaine vaccine to said subject, saidanti-cocaine vaccine comprising an injectable sterile solvent and animmunogenic amount of a compound having a formula selected from thegroup consisting of:

wherein n and m are integers from 2 to 8; whereby antisera to cocaine isproduced resulting in the suppression of the psychoactive effects tococaine.
 26. The method of claim 25, wherein said compound has thefollowing formula:

wherein n is an integer from 2 to
 8. 27. The method of claim 26, whereinin said compound n is an integer from 4 to
 6. 28. The method of claim27, wherein in said compound n is 5.